The present invention relates to separable compositions, methods, and kits for use in multiplexed assay detection of known, selected target nucleotide sequences.
The need to determine many analytes or nucleic acid sequences (for example multiple pathogens or multiple genes or multiple genetic variants) in blood or other biological fluids has become increasingly apparent in many branches of medicine. Most multi-analyte assays, such as assays that detect multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences and some require sophisticated instrumentation. Some of the known classical methods for multianalyte assays include the following:
a. The use of two different radioisotope labels to distinguish two different analytes.
b. The use of two or more different fluorescent labels to distinguish two or more analytes.
c. The use of lanthanide chelates where both lifetime and wavelength are used to distinguish two or more analytes.
d. The use of fluorescent and chemiluminescent labels to distinguish two or more analytes.
e. The use of two different enzymes to distinguish two or more analytes.
f. The use of enzyme and acridinium esters to distinguish two or more analytes.
g. Spatial resolution of different analytes, for example on arrays, to identify and quantify multiple analytes.
h. The use of acridinium ester labels where lifetime or dioxetanone formation is used to quantify two different viral targets.
As the human genome is elucidated, there will be numerous opportunities for performing assays to determine the presence of specific sequences, distinguishing between alleles in homozygotes and heterozygotes, determining the presence of mutations, evaluating cellular expression patterns, etc. In many of these cases one will wish to determine in a single reaction, a number of different characteristics of the same sample. In many assays, there will be an interest in determining the presence of specific sequences, whether genomic, synthetic, or cDNA. These sequences may be associated particularly with genes, regulatory sequences, repeats, multimeric regions, expression patterns, and the like. There will also be an interest in determining the presence of one or more pathogens, their antibiotic resistance genes, genetic subtype and the like. The need to identify and quantify a large number of bases or sequences, potentially distributed over centimorgans of DNA, offers a major challenge. Any method should be accurate, reasonably economical in limiting the amount of reagents required and provide for a highly multiplexed assay, which allows for differentiation and quantitation of multiple genes, and/or snp determination, and/or gene expression at the RNA or protein level.
The need to study differential expression of multiple genes to determine toxicologically relevant outcomes or the need to screen transfused blood for viral contaminants with high sensitivity is clearly evident. Finally, while nucleic acid sequences provide extreme diversity for situations that may be of biological or other interest, there are other types of compounds, such as proteins in proteomics that may also offer opportunities for multiplexed determinations.
There is and will continue to be comparisons of the sequences of different individuals. It is believed that there will be about one polymorphism per 1,000 bases, so that one may anticipate that there will be an extensive number of differences between individuals. By single nucleotide polymorphism (SNPs) is intended that there will be a prevalent nucleotide at the site, with one or more of the remaining bases being present in a substantially smaller percent of the population. While other genetic markers are available, the large number of SNPs and their extensive distribution in the chromosomes make SNPs an attractive target. Also, by determining a plurality of SNPs associated with a specific phenotype, one may use the SNP pattern as an indication of the phenotype, rather than requiring a determination of the genes associated with the phenotype. For the most part, the SNPs will be in non-coding regions, primarily between genes, but will also be present in exons and introns. In addition, the great proportion of the SNPs will not affect the phenotype of the individual, but will clearly affect the genotype. The SNPs have a number of properties of interest. Since the SNPs will be inherited, individual SNPs and/or SNP patterns may be related to genetic defects, such as deletions, insertions and mutations, involving one or more bases in genes. Rather than isolating and sequencing the target gene, it will be sufficient to identify the SNPs involved. In addition, the SNPs may also be used in forensic medicine to identify individuals.
Thus an assay for the differentiation and quantitation of multiple genes, and/or snp determination, and/or gene expression at the RNA or protein level, that has higher sensitivity, a large dynamic range (103 to 104-fold differences in target levels), a greater degree of multiplexing, and fewer and more stable reagents would increase the simplicity and reliability of multianalyte assays, and reduce their costs.
Holland (Proc. Natl. Acad. Sci. USA (1991) 88:7276) discloses that the exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase in PCR amplification to generate specific detectable signal concomitantly with amplification.
The TaqMan(copyright) assay is discussed by Lee in Nucleic Acid Research (1993) 21:16 3761).
White (Trends Biotechnology (1996) 14(12):478-483) discusses the problems of multiplexing in the TaqMan assay.
Marino, Electrophoresis (1996) 17:1499 describes low-stringency-sequence specific PCR (LSSP-PCR). A PCR amplified sequence is subjected to single primer amplification under conditions of low stringency to produce a range of different length amplicons. Different patterns are obtained when there are differences in sequence. The patterns are unique to an individual and of possible value for identity testing.
Single strand conformational polymorphism (SSCP) yields similar results. In this method the PCR amplified DNA is denatured and sequence dependent conformations of the single strands are detected by their differing rates of migration during gel electrophoresis. As with LSSP-PCR above, different patterns are obtained that signal differences in sequence. However, neither LSSP-PCR nor SSCP gives specific sequence information and both depend on the questionable assumption that any base that is changed in a sequence will give rise to a conformational change that can be detected. Pastinen, Clin. Chem. (1996) 42:1391 amplifies the target DNA and immobilizes the amplicons. Multiple primers are then allowed to hybridize to sites 3xe2x80x2 and contiguous to a SNP (single nucleotide polymorphism) site of interest. Each primer has a different size that serves as a code. The hybridized primers are extended by one base using a fluorescently labeled dideoxynucleoside triphosphate. The size of each of the fluorescent products that is produced, determined by gel electrophoresis, indicates the sequence and, thus, the location of the SNP. The identity of the base at the SNP site is defined by the triphosphate that is used. A similar approach is taken by Haff, Nucleic Acids Res. (1997) 25:3749 except that the sizing is carried out by mass spectrometry and thus avoids the need for a label. However, both methods have the serious limitation that screening for a large number of sites will require large, very pure primers that can have troublesome secondary structures and be very expensive to synthesize.
Hacia, Nat. Genet. (1996) 14:441 uses a high-density array of oligonucleotides. Labeled DNA samples were allowed to bind to 96,600 20-base oligonucleotides and the binding patterns produced from different individuals were compared. The method is attractive in that SNPs can be directly identified, but the cost of the arrays is high and non-specific hybridization may confound the accuracy of the genetic information.
Fan (Oct. 6-8, 1997 IBC, Annapolis Md.) has reported results of a large scale screening of human sequence-tagged sites. The accuracy of single nucleotide polymorphism screening was determined by conventional ABI resequencing.
Ross in Anal. Chem. (1997) 69:4197 discusses allele specific oligonucleotide hybridization along with mass spectrometry.
Brenner and Lerner, PNAS (1992) 89:5381, suggested that compounds prepared by combinatorial synthesis can each be labeled with a characteristic DNA sequence. If a given compound proves of interest, the corresponding DNA label is amplified by PCR and sequenced, thereby identifying the compound.
W. Clark Still, in U.S. Pat. No. 5,565,324 and in Accounts of Chem. Res., (1996) 29:155, uses a releasable mixture of halocarbons on beads to code for a specific compound on the bead that is produced during synthesis of a combinatorial library. Beads bearing a compound of interest are treated to release the coding molecules and the mixture is analyzed by gas chromatography with flame ionization detection.
U.S. Pat. No. 5,807,682 describes probe compositions for detecting a plurality of nucleic acid targets.
Methods for multiplexed detection of known, selected nucleotide target sequences are provided. In practicing the methods, target sequences are contacted with a set of electrophoretic tag (e-tag) probes under conditions that allow hybridization of the target-binding moiety of the e-tag probes to complementary target sequences.
The e-tag probe sets comprise j members, and have the form, (D, Mj)-N-Tj, where (a) D is a detection group comprising a detectable label; (b) Mj is a mobility modifier, having a particular charge/mass ratio; (c) N is a nucleotide joined to U1 in Tj through a nuclease-cleavable bond; and (d) Tj is an oligonucleotide target-binding moiety that has a sequence of nucleotides Ui connected by intersubunit linkages Bi,i+1, where i includes all integers from 1 to n, and n is sufficient to allow the moiety to specifically hybridize with a target nucleotide sequence.
After contacting a target oligonucleotide with a set of e-tag probes under hybridization conditions, the hyridized target is treated with a nuclease under conditions effective to cleave target-hybridized probes at their N-U1 linkages, thereby producing a mixture of one or more corresponding e-tag reporters of the form (D, Mj)-N, and uncleaved and/or partially cleaved probes.
Probes of the form, Mj-D-N-Tj result in the generation of e-tag reporters of the form Mj-D-N. Similarly, probes of the form, D-Mj-N-Tj, result in the generation of e-tag reporters of the form D-Mj-N.
The probes typically include a capture ligand bound to at least one nucleotide Ui, i greater than 1 in the target binding moiety of the e-tag probe. In such cases, following nuclease treatment, the mixture is exposed to a capture agent effective to bind uncleaved and/or partially cleaved probes, but not e-tag reporters, thereby either preventing the probes bound to the capture agent from electrophoretically migrating within the selected range of electrophoretic mobilities or immobilizing the probes on a solid support.
The e-tag reporters generated by the cleavage are fractionated by electrophoresis resulting in one or more electrophoretic bands. The electrophoretic mobilities of one or more electrophoretic bands are identified with each band uniquely corresponding to an e-tag reporter that is uniquely assigned to a known target sequence.
The method finds utility in multiplexed detection/analysis of targets including, but not limited to, nucleic acid detection such as sequence recognition, e.g., snp detection, transcription analysis or mRNA determinations, allelic determination, mutation determination, HLA typing or MHC determination and haplotype determination.
In one approach, the method includes the use of an e-tag probe having a capture ligand bound to at least one nucleotide U1 of the target binding moiety and capable of binding specifically to a capture agent, where i greater than 1.
Exemplary capture ligands include: (i) biotin, capable of binding specifically to capture agents such as avidin or streptavidin and (ii) an antigen, capable of binding specifically to capture agents such as an antibody or antibody fragment.
A polycation may serve as the capture agent, where the oligonucleotide has a negatively charged backbone.
In e-tag probes for use in the method, the N-U1 nuclease-cleavable bond may be a phosphodiester bond, and the nuclease-resistant bond(s) in the target-binding moiety may be one or more of thiophosphate, phosphinate, phosphoramidate, amide, and boronate linkages. Such e-tag probes may further include a capture ligand bound to at least one nucleotide Ui, i greater than 1 in the target binding moiety of the e-tag probe.